A polyethylene glycol/polycation block copolymer represented by polyethylene glycol-block-poly(L-lysine) which is a cationic block copolymer spontaneously forms a spherical micelle with an anionic macromolecule, due to the electrostatic interaction acting between the two in water as a driving power. This particle has a diameter of several tens nanometers and a core-shell structure, the core (or inner nucleus) being formed of polyion complex of cation and anion, and the shell (or outer shell) being a polyethylene glycol (which may be hereafter abbreviated as “PEG”) layer. The particle is referred to as polyion complex (PIC) micelle (see, e.g., Non-patent Reference 1 which is identified later, like other references). Thus, PIC micelles can hold anionic macromolecules in inner nuclei and are therefore expected to be capable of avoiding in vivo foreign matter recognizing mechanism due to such particle diameter as several tens nanometers and the core-shell structure. Accordingly, presently their application as a carrier (vector) of DNA which is a natural anionic macromolecule is under investigation. Although priority in developing gene vectors using such cationic block copolymers is thus clear, due to limitations on their synthesis and for other reasons, cationic block copolymers which are currently investigated do not extend beyond PEG-block-poly(L-lysine), PEG-block-poly(dimethylaminoethyl methacrylate) (see, e.g., Patent Reference 1) and PEG-block-polyethylenimine.
These PIC micelles are considerably stable under physiological conditions in general, but is actual use their stability under physiological conditions is occasionally insufficient, as exemplified by dissociation of PIC micelles under dilution after administration by intravenous injection or their interaction with serum proteins. This necessitates modification of properties of PIC micelles so that they would not dissociate but exist stably for a fixed period, until they arrive at the intended site with certainty or after their arrival. As a means to so modify properties of PIC micelles, for example, it has been proposed to improve stability of PIC micelles by introducing mercaptoalkyl groups into amino groups in a fixed proportion of L-lysine units in poly(L-lysine) segments in said PEG-block-poly-(L-lysine) to form disulfide bonds between said groups (see, e.g., Patent Reference 2).
Also as a new type, a copolymer formed by ester-amide exchange of benzyl groups in PEG-block-poly(β-benzylaspartate) with, for example, N,N-dimethylethylenediamine or the like (see, e.g. Non-patent Reference 2).
The function of polycation blocks in the micelles formed of polycation and DNA is mainly to serve as the electrostatic interaction site with the DNA, while in principle still other functions can be imparted. As one of such functions, there is proton sponge effect. Proton sponge effect refers to a phenomenon: when a polyamine of low degree of protonation is incorporated in endosomes, it absorbs hydrogen ions supplied into the endosomes by V-type ATPase one after another to prevent pH drops within the endosomes and in consequence to cause expansion of the endosomes with water infiltration accompanying rise in osmotic pressure in the endosomes, which eventually leads to destruction of the endosomes. It is expected that transfer of DNA to cytoplasm is promoted and the gene expression effectiveness is increased by this effect. This effect is seen in cations having buffer ability and, therefore, use of cations of low pKa is necessary.
On the other hand, gene expression effectiveness is considered to be affected also by stability of PIC micelles, condensed state of enclosed DNA and the like, and such factors also are presumed to be dependent on properties of individual polycation. As aforesaid, however, heretofore the kinds of studied polycation are limited and there has been no concept of simultaneous introduction of two or more kinds of polycations to allot them different functions. Under the circumstances, it was very difficult to control these factors.